The present invention relates generally to the imaging of small objects in or behind highly scattering media and more particularly to the imaging of small objects in or behind highly scattering media using microscopes.
Many small objects that we wish to observe are hidden inside or behind some kind of semi-opaque random media. Examples of such objects include subcellular components within a cell, tumors inside a breast, defects in a semiconductor, objects in tissue, etc.
When a light pulse propagates through a highly scattering medium, some of the light is multiply scattered. The multiple scattering of light reduces the intensity of the signal and increases the noise on the signal arising from the randomly scattered light. The reduction in signal and the increase in noise from multiple scattered light are the main reasons why one cannot see through an optically thick random medium. Thus, one way to enhance the quality of an image in or behind a scattering medium is to reduce the scattered light noise. The scattered light noise is typically made up of those components of the light pulse which (1) emerge from the medium at an angle relative to the angle of incidence of the light pulse on the medium; and/or (2) take the longest period of time to emerge from the medium.
In U.S. Pat. No. 5,140,463, inventors Yoo et al., which issued Aug. 18, 1992, and which is incorporated herein by reference, there is disclosed a method and apparatus for improving the quality of an image of an object hidden inside a highly scattering, semi-opaque, disordered medium using space gate imaging or time gate imaging or space time gate imaging. According to the patent, in space gate imaging, a small segment of the object is illuminated at a time. The scattered light is passed through a spatial noise filter. On the image plane, an aperture is open at the position of the image segment which corresponds to the segment of the illuminated object. A full image is obtained by scanning the object, segment by segment, and simultaneously recording the signal at the corresponding image segment. In time gate imaging, the unscattered (i.e. ballistic) portion of the pulse which contains the information of the image is temporally separated from the other (i.e. scattered) portions which contain the noise using an ultrafast laser pulse and temporal gating devices. In space-time gating, the two techniques are combined to produce an image with a much higher signal to noise ratio. The time separation between the ballistic and scattered light may be increased by increasing thickness of random medium or by introducing small scatters into the random medium so as to make the medium more random. The signal to noise ratio can also be increased by making the random medium less random (so that there will be less scattered light). In addition, the signal to noise ratio can be increased by introducing an absorbing dye into the medium or by using a wavelength for the light which is in the absorption spectrum of the random medium or by making the medium more ordered (i.e. less random) or by using a pair of parallel polarizers.
In U.S. patent application Ser. No. 07/920,193 and U.S. Pat. No. 5,371,368, inventors Robert R. Alfano et al., which was filed Jul. 23, 1992, and which is incorporated herein by reference, there is disclosed a system for imaging an object in or behind a highly scattering medium. According to the patent application, the system includes a laser for transilluminating the highly scattering medium. The light emerging from the highly scattering medium consists of a ballistic component, a snake-like component and a diffuse component. In one embodiment of the invention, a Kerr gate is used to temporally gate the light exiting the transilluminated medium. The Kerr gate, which is controlled by a pump beam of light, opens for an appropriately short period of time to permit the ballistic and snake components of the light exiting the medium to pass therethrough for imaging and then closes to prevent the diffuse component of the light from passing therethrough. In another embodiment of the invention, a 4F Fourier gate is additionally used to spatially gate the light exiting the transilluminated medium. The 4F Fourier gate improves image quality by filtering out the components of light exiting the medium at large angles, i.e., the diffuse component. The Kerr gate and the 4F Fourier gate may be combined by placing the Kerr gate at the 2F spectral plane and by gating only that portion of the Kerr gate situated at the focal point of the 4F Fourier system.
In U.S. patent application Ser. No. 07/927,566 which is abandoned on Jan. 28,1994, inventors Robert R. Alfano et al., which was filed Aug. 10, 1992, and which is incorporated herein by reference, there is disclosed a technique for forming an image of an object located in or behind a scattering medium. In one embodiment, the object is made luminescent, and the luminescent light is selected for imaging while the illuminating light is filtered out. The quality of the image can be further improved by selecting the portion of the luminescence spectrum that is strongly absorbed by the scattering medium.
Neither the aforementioned patent nor the aforementioned patent applications are directed to the imaging of objects in highly scattering media using microscopes.
Microscopes are optical devices commonly used to examine small objects at large magnifications. In its simplest form, a microscope comprises an objective and an eyepiece. The objective typically comprises one or more lenses which are used to form a real, inverted and much enlarged image of an object on the focal plane of the eyepiece. The eyepiece (or ocular) acts as a collimator so that one looking into it sees a virtual image of the object, subtending a wide angle. Many microscopes include a light source for illuminating or transilluminating the medium in which the object is located.
Ordinary microscopes cannot obtain clear images of objects hidden inside or behind highly scattering cloudy media. Scanning confocal microscopes, however, have been used to locate some objects in highly scattering media and have been used with some success to form images of some objects in highly scattering media. Moreover, by combining the techniques of dye staining and multiple photon nonlinear optical excitation with confocal microscopy, improved images of objects inside highly scattering media have been obtained. Time-resolved techniques have also been used with confocal microscopy to improve the images of objects located inside highly scattering media.
Publications of interest include F. G. Smith and J. H. Thomson, "Optics," 2nd Ed., Manchester Physics Series, John Wiley, pp. 214-217 (1988); "Introduction to Optics," F. Pedrotti and L. Pedrotti, Prentice Hall (2nd), pp. 135-138 (1993); R. W. Ditchburn, "Light," John Wiley, 2nd Ed., pp. 290-295 (1963); and Kempe and Rudolph, "Analysis of confocal microscope under ultrashort light pulse illumination," JOSA A, Vol. 10, pp. 240-245 (1993).